Four new species of Asian horned toads (Anura, Megophryidae, Megophrys) from southern China

Abstract Recent phylogenetic analysis encompassing multilocus nuclear-gene and matrilineal mtDNA genealogy has revealed a series of cryptic species of the subgenus Panophrys within genus Megophrys from southern and eastern China. This study demonstrates that the Panophrys specimens from the hilly areas among Guangdong, Guangxi and Hunan can be morphologically distinguished from all recognized congeners, thereby providing additional supports for the recognitions of four new species of Panophrys, namely Megophrys (Panophrys) mirabilis Lyu, Wang & Zhao, sp. nov. from northeastern Guangxi, Megophrys (Panophrys) shimentaina Lyu, Liu & Wang, sp. nov. from northern Guangdong, and Megophrys (Panophrys) xiangnanensis Lyu, Zeng & Wang, sp. nov. and Megophrys (Panophrys) yangmingensis Lyu, Zeng & Wang, sp. nov. from southern Hunan. The descriptions of these species take the number of Megophrys species to 101, 46 of which belong to the subgenus Panophrys.


introduction
The Asian horned toad genus Megophrys Kuhl & Van Hasselt, 1822 within the family Megophryidae Bonaparte, 1850, is a typical representative for Oriental fauna, spreading throughout southern China, southern and eastern Himalayas, across Indochina to islands of the Sunda Shelf and the Philippines Liu et al. 2018;Frost 2020). Although morphological identifications on Megophrys species are not easy (Li et al. 2014;Liu et al. 2018), with the progress in integrative taxonomy, a large number of new species have been recognized in the last decade, and takes the species number of genus Megophrys sensu lato to 97 (Frost 2020).
During our herpetological surveys in the hilly areas among Guangdong, Guangxi and Hunan, southern China (Fig. 1), a series of specimens of horned toads were collected. These specimens morphologically belong to genus Megophrys but could not be assigned to any recognized species by the combinations of characteristics. Furthermore, the phylogenetic analysis encompassing multilocus nuclear-gene and matrilineal mtD-NA genealogy conducted by Liu et al. (2018) has indicated that these specimens should be regarded as four cryptic species of the subgenus Panophrys, i.e., M. sp29 from northern Guangdong, M. sp25 from northeastern Guangxi, and M. sp2 and M. sp28 from southwestern Hunan. In this study, as a follow-up work on this series of specimens, we provide the additional morphological comparisons and descriptions to substantiate the recognition of these four cryptic species of Panophrys from southern China.

Materials and methods
Taxonomic system. The higher systematics of Asian horned toads has been in intensive debates for decades (Delorme et al. 2006;Frost et al. 2006;Li and Wang 2008;Chen et al. 2017;Deuti et al. 2017;Mahony et al. 2017;Liu et al. 2018;. In this study, not involving in the controversy of generic relationship in subfamily Megophryinae, we followed the most recent taxonomic arrangement Liu et al. 2018;Frost 2020), in which the genus Megophrys is considered to include seven subgenera: Atympanophrys Tian & Hu, 1983, Brachytarsophrys Tian & Hu, 1983, Megophrys, Ophryophryne Boulenger, 1903, Pelobatrachus Beddard, 1908, Panophrys Rao & Yang, 1997, and Xenophrys Günther, 1864. Since the subgenus Panophrys has been unanimously considered as a monophyletic group that is significantly divergent from other subgenera, we perform the analyses and comparisons on the undescribed specimens with Panophrys congeners in this study.
Phylogeny. Two mitochondrial genes, namely partial 16S ribosomal RNA gene (16S) and partial cytochrome C oxidase 1 gene (CO1), were used for phylogenetic analysis. All sequences were attained from GenBank, encompassing 17 samples of the unnamed species (originally submitted by Liu et al. 2018) and 40 samples from 40 recognized Panophrys congeners. Besides, two samples of subgenus Xenophrys were incorporated into our dataset as out-groups. Detailed information of these materials is given in Table 1. DNA sequences were aligned by the Clustal W algorithm with default parameters (Thompson et al. 1997) and trimmed with the gaps partially deleted in MEGA 6 (Tamura et al. 2013). Two gene segments, 632 base pairs (bp) of CO1 and 541 bp of16S, were concatenated seriatim into a 1173-bp sequence, and were further tested in jmodeltest v2.1.2 with Akaike and Bayesian information criteria, all resulting the best-fitting nucleotide substitution models of GTR+I+G. Sequenced data was analyzed using Bayesian inference (BI) in MrBayes 3.2.4 (Ronquist et al. 2012). Two independent runs were conducted in a BI analysis, each of which was performed for 10,000,000 generations and sampled every 1000 generations with the first 25% samples were discarded as burn-in, resulting a potential scale reduction factor (PSRF) of < 0.005. Mean genetic distances of 16S gene between and within species were calculated in MEGA 6 using the uncorrected p-distance model.
Bioacoustics. Advertisement calls of the unnamed species were recorded in the field by a SONY PCM-D50 digital sound recorder. The sound files in wave format were sampled at 48 kHz with 24 bits in depth. Raven pro 1.5 (Cornell Lab of Ornithology, 2003Ornithology, -2014 was used to output the spectrograms and to measure interrelated parameters with Fast Fourier transform of 256 points and a 50% overlap. The following measurements were performed: call/note duration (the difference between begin time and end time for a selected call/note), notes per call, inter-note intervals (the difference between end time for a selected note and begin time for the next selected note), peak frequency (the frequency at which peak power occurs within the selected call), high frequency (the highest frequency of the selected call), low frequency (the lowest frequency of the selected call), bandwidth 90% (the difference between the 5% and 95% frequencies of a selected call).
Morphology. Thirty-six unnamed specimens from the hilly areas among Guangdong, Guangxi and Hunan, southern China were examined, 17 of which have been used in the phylogenetic analysis. All examined specimens were fixed in 10% buffered formalin and later transferred to 70% ethanol. All studied specimens are deposited in The Museum of Biology, Sun Yat-sen University (SYS), and Chengdu Institute of Biology, Chinese Academy of Sciences (CIB), China.
External measurements were made for the unnamed specimens with digital calipers (Neiko 01407A Stainless Steel 6-Inch Digital Caliper, USA) to the nearest 0.1 mm. Mean and standard deviation (SD) were calculated in R 3.3.2 (R Core Team 2016). These measurements were as follows:

ED
eye diameter (from the anterior corner of the eye to posterior corner of the eye); FTL foot length (from distal end of shank to the tip of digit IV); HDL head length (from tip of snout to the articulation of the jaw); HDW head width (head width at the commissure of the jaws); HND hand length (from the proximal border of the outer palmar tubercle to the tip of digit III); IND internasal distance (distance between nares); IOD interorbital distance (minimum distance between upper eyelids); RAD radio-ulna length (from the flexed elbow to the proximal border of the outer palmar tubercle); SNT snout length (from tip of snout to the anterior corner of the eye); SVL snout-vent length (from tip of snout to posterior margin of vent); TD tympanum diameter (horizontal diameter of tympanum); TED tympanum-eye distance (from anterior edge of tympanum to posterior corner of the eye); TIB tibial length (from the outer surface of the flexed knee to the heel).
Sex was determined by secondary sexual characters, i.e., the presence of vocal sac, nuptial pads/spines in males (Fei et al. 2016).
Morphological characters of all 42 recognized congeners of subgenus Panophrys for comparisons were based on the examination of museum specimens listed in Appendix I and on information available in the literature (Table 2).
(Pa.) wushanensis 3.6 3.6 3.6 2.8 8.1 2.1 4.0 3.9 2.6 4.9 3.4 3.2 3.6 3.9 3.8 3.6 3.0 3.6 7.7 4.3 3.2 3.0 3.2 2.4 3.4 4.5 4.0 6.0 3.2 3.7 5.0 6.0 4.3 3.7 5.0 3.8 4.5 2.6 4.5 1.7 3.9 3.9 / 57 M. 1.00) and almost have no molecular divergences (p-distances 0.0), which was defined as a cryptic species Megophrys sp25 in Liu et al. (2018); this population can be further distinguished from all recognized and undescribed species by a combination of distinctive morphological characters (see Taxonomic accounts below). Therefore, the population from Huaping Nature Reserve represents a separately evolving lineage, and is described as a new species, Megophrys (Panophrys) mirabilis sp. nov. The samples from Shimentai Nature Reserve, Guangxi (samples ID 5-8 in Table 1), are grouped into a monophyletic clade with strong node supports (BPP 1.00) and almost have no molecular divergences (p-distances 0.0), which was defined as a cryptic species Megophrys sp29 in Liu et al. (2018); samples (ID 14-17 in Table 1) from Mt Yangming, Hunan, are clustered into a monophyletic clade with strong node supports (BPP 1.00) and have small molecular divergences (p-distances 0.3), which was defined as a cryptic species M. sp28 in Liu et al. (2018). These two populations are sister taxa to each other with significant genetic divergences (p-distances 4.1), and can be distinguished from all congeners by a combination of distinctive morphological characters (see Taxonomic accounts below). Therefore, the populations from Shimentai Nature Reserve and Mt Yangming represent two separately evolving lineage, and are described as new species, Megophrys (Panophrys) shimentaina sp. nov. and Megophrys (Panophrys) yangmingensis sp. nov., respectively.
The other samples from Mt Yangming, Hunan (samples ID 9-13 in Table 1), cluster into a monophyletic clade with strong node supports (BPP 1.00) and almost have no molecular divergences (p-distances 0.0), which was defined as a cryptic species Megophrys sp2 in Liu et al. (2018). This clade is conspicuously distant from the sympatric species Megophrys (Panophrys) yangmingensis sp. nov. in phylogeny. Furthermore, this population can be distinguished from all congener species by a combination of distinctive morphological characters (see Taxonomic accounts below). Therefore, this population from Mt Yangming represents a separately evolving lineage, and is described as a new species, Megophrys (Panophrys) xiangnanensis sp. nov. Paratypes. Three adult specimens from the same locality as the holotype: male SYS a002192 and female SYS a002193 collected on 10 July 2013 by Jian Zhao and Yu-Long Li; female SYS a002289 collected on 9 September 2013 by Zu-Yao Liu. Etymology. The specific epithet mirabilis means marvelous, referring to its distinctive habitus and color pattern of this species within the subgenus Panophrys.
(1) Body size relatively large, SVL 55.8-61.4 mm (N = 2) in adult males and SVL 68.5-74.8 (N = 2) mm in adult females; (2) snout rounded in dorsal view; (3) internasal distance smaller than interorbital distance; (4) tympanum clear, moderate size, TD/ED 0.49-0.63; (5) absence of vomerine ridge and vomerine teeth; (6) tongue small, majorly attached to the mandible, free margin small and rounded, not notched behind; (7) hindlimbs slender, heels overlapping and tibio-tarsal articulation reaching forward at the central eye; (8) fingers with distinct lateral fringes, presence of indistinct subarticular tubercles at the bases; (9) toes with distinct lateral fringes and rudiment of webs, presence of indistinct subarticular tubercles at the bases; (10) presence of slightly large horn-like tubercle at the edge of upper eyelid; (11) dorsal skin smooth with granules, (12) skin on flanks flabby, with spiny tubercles; (13) supratympanic fold distinct, with dense tubercles, forming an extremely swollen large shoulder gland above insertion of arm; (14) grayish brown above, tinged with blue in males, but dorsum of head and body reddish brown in females; (15) ventral surface of throat and chest with grayish blue latticed patches and black spots in males, but with orange latticed patches and black spots in females; (16) presence of underdeveloped nuptial pads on the dorsal surface of the first finger in adult males.
Comparison. Megophrys (Panophrys) mirabilis sp. nov. can be easily distinguished from all recognized congeners, by having a small tongue, majorly attached to the mandible, flank skin flabby with spiny tubercles, and supratympanic fold with dense tubercles forming an extremely swollen large shoulder gland above insertion of arm.
Further, detailed comparative data of Megophrys (Panophrys) mirabilis sp. nov. with 42 recognized congeners of Panophrys are given in Table 4.
Five Panophrys species were previously recorded from the hilly areas among Guangdong, Guangxi, and Hunan, namely  Description of holotype. Adult male. Body size large, SVL 61.4 mm; head width slightly larger than head length, HDW/HDL 1.02; snout rounded in dorsal view, projecting, sloping backward to mouth in profile, protruding well beyond margin of lower jaw; top of head flat; eyes large, ED 0.31 of HDL, pupil vertical; nostril oblique-ovoid; canthus rostralis well developed; loreal region slightly oblique; internasal distance smaller than interorbital distance; tympanum clear, TD/ED 0.49; large ovoid choanae at the base of the maxilla; absence of vomerine ridge and vomerine teeth; tongue small, majority attached at the mouth, margin rounded, not notched behind; absence of vocal sac.
Radio-ulna length 0.26 of SVL and hand 0.28 of SVL; hand without webs, fingers with distinct lateral fringes, relative finger length II < I < IV < III; tips of fingers slightly dilated, round; one indistinct subarticular tubercle at the bases of each finger; metacarpal tubercles indistinct, the inner one observably enlarged and the outer one smaller; presence of underdeveloped nuptial pad on the dorsal surface of the first finger, without nuptial spines. Hindlimbs slender, tibio-tarsal articulation reaching forward at the central eye when hindlimb is stretched along the side of the body; heels overlapping when the flexed hindlimbs are held at right angles to the body axis; tibia length 0.47 of SVL and foot length 0.71 of SVL; relative toe length I < II < V < III < IV; tips of toes round and slightly dilated; toes with narrow lateral fringes and rudiment of webs; one indistinct subarticular tubercle at the bases of each toe; inner metatarsal tubercle long ovoid and the outer one absent.
Dorsal skin smooth with sparse granules; flanks flabby with spiny tubercles; distinct supratympanic fold curving postero-ventrally from posterior corner of eye to a level above insertion of arm; small tubercles arranged from above the nostril, along the canthus rostralis, edge of upper eyelid and supratympanic fold, to the posterior margin of temporal region; a distinct horn-like prominent tubercle on the edge of upper eyelid; a discontinuous X-shaped ridge with several short ridges on two sides on the back; transverse skin ridges on the dorsal shank and thigh; ventral surface smooth; several tubercles on posterior hindlimbs; small pectoral gland closer to axilla; a single large femoral gland on rear of thigh.
Coloration. Grayish brown above in life; an dark interorbital triangle with light colored center and edge; a dark X-shaped making with light edge on the central of dorsum; dark brown transverse bands on forearms and hindlimbs; supratympanic fold light gray; dark vertical band below the eye; iris grayish brown; ventral surface grayish white; throat and chest with grayish blue latticed patches and black spots; ventral hands and feet grayish white, tips of digits creamy white, metacarpal tubercle and metatarsal tubercle grayish white; pectoral gland and femoral gland white.
Variations. Measurement data of type series are listed in Table 5. All paratypes are similar to the holotype. Females (SVL 68.5-74.8 mm) are significantly larger than males (SVL 55.8-61.4 mm). Dorsal surfaces reddish brown and ventral surfaces with orange latticed patches and black spots in females SYS a002193, 2289.
Distribution and ecology. Currently, Megophrys (Panophrys) mirabilis sp. nov. is only known from Huaping Nature Reserve, northeastern Guangxi. The individuals were found on shrubbery branches near trail paths between elevations of 1300-1330 m a.s.l. from June to September. Males were not calling when found, but the collected female specimens bear mature yellowish oocytes. Tadpoles have not been found and ecological information remains unknown.  Etymology. The specific epithet shimentaina refers to its type locality, Shimentai Nature Reserve.
(1) Body size small, SVL 28.0-30.6 (28.9 ± 0.9, N = 12) mm in adult males; (2) snout rounded in dorsal view; (3) tympanum clear, TD/ED 0.57-0.66; (4) presence of weak vomerine ridge and vomerine teeth; (5) margin of tongue rounded, not notched behind; (6) hindlimbs slender, heels overlapping and tibio-tarsal articulation reaching forward between tympanum to anterior corner of eye; (7) tibia 0.44-0.53 of SVL and foot 0.62-0.76 of SVL; (8) fingers with narrow lateral fringes, presence of indistinct subarticular tubercles at the bases; (9) toes with narrow lateral fringes and rudiment of webs, absence of subarticular tubercle; (10) presence of a small horn-like tubercle at the edge of upper eyelid; (11) presence of tiny, barely visible, black to dark brown spines on the whole dorsal skin, flanks, dorsal limbs, the region around cloaca, and rear of hindlimbs; (12) dorsal skin rough, a discontinuous "/ \"-shaped ridge with two discontinuous dorsolateral ridges on two sides on the back; (13) several large warts on the flanks; (14) supratympanic fold distinct and white, with tiny spines; (15) light brown above, a dark brown stripe on each upper eyelid; (16) single subgular vocal sac in males; (17) weak nuptial pads with serried olive nuptial spines, on the dorsal surface of the first and second fingers in adult males.
Comparison. Comparative data of Megophrys (Panophrys) shimentaina sp. nov. with M. (Pa.) mirabilis sp. nov. and 42 recognized congeners of Panophrys are given in Table 4. Description of holotype. Adult male. Body size small, SVL 28.4 mm; head width slightly smaller than head length, HDW/HDL 0.95; snout rounded in dorsal view, projecting, sloping backward to mouth in profile, protruding well beyond margin of lower jaw; top of head flat; eyes large, ED 0.33 of HDL, pupil vertical; nostril obliqueovoid; canthus rostralis well developed; loreal region slightly oblique; internasal distance slightly larger than interorbital distance; tympanum clear, in medium size, TD/ ED 0.61; large ovoid choanae at the base of the maxilla; presence of weak vomerine ridge and vomerine teeth; margin of tongue rounded, not notched behind; presence of a single subgular vocal sac, a pair of slit-like openings at posterior of jaw.
Radio-ulna length 0.22 of SVL and hand 0.26 of SVL; hand without webs, fingers with narrow lateral fringes, relative finger length I ≈ II < IV < III; tips of fingers slightly dilated, round; one indistinct subarticular tubercle at the bases of each finger; inner metacarpal tubercle observably enlarged and the outer one smaller; nuptial pads with serried olive nuptial spines on the dorsal surface of the first and second fingers. Hindlimbs slender, tibio-tarsal articulation reaching forward to the posterior corner of eye when hindlimb is stretched along the side of the body; heels overlapping when the flexed hindlimbs are held at right angles to the body axis; tibia length 0.47 of SVL and foot length 0.67 of SVL; relative toe length I < II < V < III < IV; tips of toes round and slightly dilated; toes with distinct lateral fringes and rudiment of webs, without subarticular tubercle; inner metatarsal tubercle long ovoid and the outer one absent.
Dorsal skin rough; numerous granules densely arranged on the top of head, loreal region, lips, temporal region, dorsal body, flanks and dorsal limbs; several tubercles on upper eyelid, including a horn-like prominent tubercle on the edge; all granules and tubercles bearing tiny, barely visible spines; clear supratympanic fold with tiny spines, curving postero-ventrally from posterior corner of eye to a level above insertion of arm; tubercles and granules forming discontinuous "/ \"-shaped ridge and two discontinuous dorsolateral ridges on two sides at the central back; large tubercles and warts on the flanks; ventral surface smooth; several granules bearing black spines on the region around cloaca and rear of hindlimbs; small pectoral gland closer to axilla; a single large femoral gland on rear of thigh.
Coloration. Light brown above in life; a dark brown stripe on dorsal surface of each eye; narrow dark brown transverse bands on forearms and hindlimbs; supratympanic fold white; dark vertical band below the eye; iris reddish brown; all spines black or dark brown; ventral surface pale; throat flesh color; scarlet spots on the chest; a large white blotch on the belly; a pair of lateroventral longitudinal broad black stripes with several white tubercles on two sides; ventral limbs flesh color with white spots; ventral hands and ventral feet brown, tips of digits pale brown; metacarpal tubercle and metatarsal tubercle reddish; pectoral gland and femoral gland white.
Variations. Measurement data of type series are listed in Table 6. All paratypes are extremely similar to the holotype but SYS a002082 has an "X" pattern on its back. Distribution and ecology. Currently, Megophrys (Panophrys) shimentaina sp. nov. is known only from Shimentai Nature Reserve, northern Guangdong. This toad is uncommon in its distribution areas. All individuals were found from two slowly flowing mountain streams between elevations of 210-500 m a.s.l. Males call on plant leaves from April to August, suggesting their breeding season corresponds to this period. Females and tadpoles have not been found.
Vocalization. The advertisement calls of Megophrys (Panophrys) shimentaina sp. nov. were recorded from four males at 18-20 °C air temperature on 27 April 2016. Thirty calls with 96 notes are measured and the spectrograms are shown in Fig. 6A. The advertisement call is made up of 3.8 Paratypes. Eleven adult specimens, female SYS a002874 and males SYS a002876/ CIB 116072 and SYS a002878-2886, collected at the same time from the same locality as the holotype.
Etymology. The specific epithet xiangnanensis is an adjective derived from Chinese Pinyin Xiāng Nán, which means southern Hunan, for the distribution area of this species.
(1) Moderate body size, SVL 38.6-42.0 mm (40.3 ± 1.3, N = 11) in adult males and SVL 44.4 mm in adult female; (2) snout rounded in dorsal view; (3) tympanum clear, TD/ED 0.38-0.49; (4) presence of weak vomerine ridge, absence of vomerine teeth; (5) margin of tongue rounded, not notched behind; (6) hindlimbs slender, heels just meeting and tibio-tarsal articulation reaching forward between eye and tympanum; (7) tibia 0.41-0.46 of SVL and foot 0.57-0.62 of SVL; (8) fingers without lateral fringes, presence of distinct subarticular tubercles at the bases; (9) toes with relatively wide lateral fringes and rudiment of webs, presence of distinct subarticular tubercles at the bases; (10) presence of small horn-like tubercle at the edge of upper eyelid; (11) dorsal skin smooth with sparse granules, a discontinuous X-shaped ridge with two discontinuous dorsolateral ridges on two side on the back; (12) sparse tubercles on the flanks; (13) supratympanic fold light colored; (14) single subgular vocal sac in males; (15) presence of nuptial pads on the dorsal surface of the first and second fingers in adult males. Description of holotype. Adult male. Moderate body size, SVL 40.9 mm; head width slightly larger than head length, HDW/HDL 1.02; snout rounded in dorsal view, projecting, sloping backward to mouth in profile, protruding well beyond margin of lower jaw; top of head flat; eyes large, ED 0.41 of HDL, pupil vertical; nostril oblique-ovoid; canthus rostralis well developed; loreal region slightly oblique; internasal distance slightly larger than interorbital distance; tympanum clear, TD/ED 0.44; large ovoid choanae at the base of the maxilla; presence of weak vomerine ridge, ab-sence of vomerine teeth; margin of tongue rounded, not notched behind; presence of a single subgular vocal sac, a pair of slit-like openings at posterior of jaw.
Radio-ulna length 0.22 of SVL and hand 0.23 of SVL; hand without webs, fingers without lateral fringes, relative finger length I < II < IV < III; tips of fingers slightly dilated, round; one distinct subarticular tubercle at the bases of each finger; inner metacarpal tubercle observably enlarged and the outer one smaller; a single nuptial pad on the dorsal surface of the first and second fingers. Hindlimbs slender, tibio-tarsal articulation reaching forward between eye and tympanum when hindlimb is stretched along the side of the body; heels just meeting when the flexed hindlimbs are held at right angles to the body axis; tibia length 0.42 of SVL and foot length 0.58 of SVL; relative toe length I < II < V < III < IV; tips of toes round and slightly dilated; toes with relatively wide lateral fringes and rudiment of webs; one distinct subarticular tubercle at the bases of each toe; inner metatarsal tubercle long ovoid and the outer one absent.
Dorsal skin smooth with sparse granules; sparse tubercles on the flanks; a horn-like prominent tubercle on the edge; clear supratympanic fold curving postero-ventrally from posterior corner of eye to a level above insertion of arm; a discontinuous Xshaped ridge and two discontinuous dorsolateral ridges on two sides at the central back; sparse tubercles on the dorsal shank and thigh; ventral surface smooth; several tubercles on posterior hindlimbs; small pectoral gland closer to axilla; a single large femoral gland on rear of thigh.
Coloration. Yellowish brown above in life; a dark interorbital triangle with light colored center and edge; a dark X-shaped making with light edge on the central of dorsum; dark brown transverse bands on forearms and hindlimbs; supratympanic fold light colored; dark vertical band below the eye; iris light brown with net-like stripes; throat and anterior chest reddish gray; a longitudinal stripe on the throat; a large white blotch with scarlet spots on the belly; one pair of lateroventral longitudinal broad reddish stripes on two sides; ventral limbs flesh color; ventral hands purplish, tips of fingers pale-grey, metacarpal tubercle reddish; ventral feet purplish brown, tips of fingers pale grey, metatarsal tubercle reddish; pectoral gland and femoral gland white.
Variations. Measurement data of type series are listed in Table 7. All paratypes are similar to the holotype. Female (SVL 44.4 mm) are slightly larger than males (SVL 38.6-42.0 mm).
Distribution and ecology. Megophrys (Panophrys) xiangnanensis sp. nov. is currently known only from Mt Yangming, southwestern Hunan. This toad inhabits areas near slowly flowing mountain streams surrounded by moist subtropical secondary evergreen broadleaf forests between elevations of 900-1400 m a.s.l. Males call from May to July, and during this time the males bear nuptial pads. Only one female individual was found, and tadpoles and other ecological information remain unknown.
Vocalization. The advertisement calls of Megophrys (Panophrys) xiangnanensis sp. nov. were recorded from the Holotype at 16 °C air temperature on 12 June 2014. Four calls with 98 notes are measured and the spectrograms are shown in Fig. 6B. The advertisement call is made up of 24.5 ± 4.7 (17-29, N = 4) continuous click notes. Each call lasts 9.46 ± 1.77 s (6.39-10.53 s, N = 4) and each note lasts 151 ± 12 ms (113- fringes, presence of distinct subarticular tubercles at the bases; (9) toes with lateral fringes and rudiment of webs, presence of subarticular tubercles at the bases; (10) presence of small horn-like tubercle at the edge of upper eyelid; (11) dorsal skin rough with sparse granules, a discontinuous X-shaped ridge with two discontinuous dorsolateral ridges on two side on the back; (12) sparse tubercles on the flanks; (13) orange-brown or light brown above, a dark interorbital triangle with light colored center and edge, a dark X-shaped making with light edge on the central of dorsum; (14)  Description of holotype. Adult male. Body size moderate, SVL 35.1 mm; head width slightly larger than head length, HDW/HDL 1.01; snout rounded in dorsal view, projecting, protruding well beyond margin of lower jaw; top of head flat; eyes large, ED 0.43 of HDL, pupil vertical; nostril oblique-ovoid; canthus rostralis well developed; loreal region slightly oblique; internasal distance slightly larger than interorbital distance; tympanum clear, TD/ED 0.43; large ovoid choanae at the base of the maxilla; presence of weak vomerine ridge, absence of vomerine teeth; margin of tongue rounded, not notched behind; presence of a single subgular vocal sac, a pair of slit-like openings at posterior of jaw.
Radio-ulna length 0.24 of SVL and hand 0.23 of SVL; hand without webs, fingers without lateral fringes, relative finger length II < I < IV < III; tips of fingers slightly dilated, round; one distinct subarticular tubercle at the bases of each finger; inner metacarpal tubercle observably enlarged and the outer one smaller; villiform black nuptial spines on the dorsal surface of the first and second fingers. Hindlimbs slender, tibio-tarsal articulation reaching forward at the anterior corner of eye when hindlimb is stretched along the side of the body; heels overlapping when the flexed hindlimbs are held at right angles to the body axis; tibia length 0.51 of SVL and foot length 0.67 of SVL; relative toe length I < II < V < III < IV; tips of toes round and slightly dilated; toes with lateral fringes and rudiment of webs; one subarticular tubercle at the bases of each toe; inner metatarsal tubercle long ovoid and the outer one absent.
Dorsal skin rough with sparse granules; sparse tubercles on the flanks and hindlimbs; several tubercles on upper eyelid, including a horn-like prominent tubercle on the edge; clear supratympanic fold curving postero-ventrally from posterior corner of eye to a level above insertion of arm; a discontinuous X-shaped ridge and two discontinuous dorsolateral ridges on two sides at the central back; four transverse skin ridges on the dorsal shank and thigh; ventral surface smooth; several granules on posterior hindlimbs; small pectoral gland closer to axilla; a single large femoral gland on rear of thigh.
Coloration. Orange-brown above in life; a triangular making with light edge between eyes; a dark X-shaped making with light edge on the central of dorsum; supratympanic fold light brown; dark vertical band below the eye; iris orangebrown; throat and anterior chest purplish brown; belly dark gray with a large white blotch on the central; ventral limbs purplish; ventral hands reddish brown with dark stripes, tips of fingers pale-grey, metacarpal tubercle reddish; ventral feet purplish, tips of fingers pale-grey, metatarsal tubercle reddish; pectoral gland and femoral gland white.
Variations. Measurement data of type series are listed in Table 8. All paratypes are similar to the holotype. The single female (SVL 45.2 mm) are distinctly larger than males (SVL 33.2-37.1 mm), while with relatively shorter hindlimbs. Dorsal surfaces lighter brown in SYS a002877, 2888-2889, 2891-2892.
Distribution and ecology. Currently, Megophrys (Panophrys) yangmingensis sp. nov. is only known from Mt Yangming, southwestern Hunan. This toad inhabits near flowing mountain streams over 1300 m a.s.l. Males call from early June to early September. Males found in early June bear well developed nuptial spines, while the spines are absent in males found in early September, suggesting the breeding season of this toad is before September. Only one female was found, and tadpoles and more ecological information remain unknown.

Discussion
The phylogenetic analysis encompassing multilocus nuclear-gene and matrilineal mtDNA genealogy ) has revealed 41 cryptic species within the subgenus Panophrys. Subsequently, eight of them were described as seven new species (Li et al. 2018;Wang et al. 2019a, b). It is worth noting that the cryptic species M. sp6 and M. sp7 revealed based on molecular data were suggested to be the same species and is described as M. (Pa.) nanlingensis after detailed morphological examination (Wang et al. 2019a). In our present study, we propose four new species, on the basis of detailed morphological evidences combined with previous phylogenetic data. There are 29 undescribed cryptic species remaining according to Liu et al. (2018), nevertheless, the recognitions from molecular data still require validation from detailed morphological characteristics to substantiate.
The genus Panophrys was established by Rao and Yang (1997) but was controversially considered as a subgenus or synonymy of Xenophrys or Megophrys by different subsequent morphological researches (Dubois and Ohler 1998; Delorme et al. 2006;Li and Wang 2008;Fei et al. 2009). Based on multilocus nuclear-gene and matrilineal mtDNA genealogy, three recent studies have revealed highly similar phylogenetic relationships within Megophryinae, which is unanimously considered to contain the following monophyletic groups: Pelobatrachus, Megophrys, Xenophrys, Panophrys, Brachytarsophrys, Ophryophryne, Atympanophrys (Chen et al. 2017;Mahony et al. 2017;Liu et al. 2018). However, the taxonomic proposals for these groups are in conflict by different authors. Chen et al. (2017) considered that subfamily Megophryinae is valid and composed of five genera: Atympanophrys, Brachytarsophrys, Megophrys, Ophryophryne and Xenophrys (including Panophrys as a subgenus). Mahony et al. (2017) treated the entire subfamily Megophryinae as a single genus Megophrys with containing seven subgenera (corresponding to the seven molecularly resolved clades). To resolve these conflicts,  suggested to elevate the seven monophyletic subgenera to genus levels, which fulfills the following three criteria to be descriptively useful: reasonably compact, monophyletic, and ecologically, morphologically or biogeographically distinct (Gill et al. 2005). Li et al.'s suggestion was based on the review of Brachytarsophrys, which shows significant differences against other groups. Therefore, the recognition of genus Brachytarsophrys must be accepted, while further supported evidences for other genera are needed.